Influence of amine-modified poly(vinyl alcohol)s on vibrating-membrane nebulizer performance and lung toxicity.

A suitable aerosol droplet size and formulation output rate is essential for the therapy of lung diseases under application of nebulizers. The current study investigated the potential of amine-modified poly(vinyl alcohol)s as excipients for inhalation delivery. A change of conductivity (effective at <0.1mg/ml) and viscosity (effective at >0.1mg/ml) of samples that were supplemented with charge-modified polymers had a significant influence on the generated droplet size (shift from ~8 to ~4 µm) and formulation throughput rate (shift from ~0.2 to ~1.0 g/min), where polymers with a higher amine density (and molecular weight) showed an elevated activity. Biocompatibility assessment of polymers in A549 cells and an isolated lung model resulted in cell lysis and lung edema formation dependent on the type (degree of amine substitution) and dose of polymer applied. Suitable compositions and concentrations of amine-modified poly(vinyl alcohol)s were identified with respect to an optimized nebulizer performance and acceptable biocompatibility. Charge-modified polymers represent novel excipients with potential to improve inhalation therapy.

Prolonged vasodilatory response to nanoencapsulated sildenafil in pulmonary hypertension.

Direct vasodilator delivery to the airways enables a selective therapy of pulmonary hypertension (PH). However, short-term effects of the applied medication require multiple daily inhalations. Controlled release formulations (polymeric nanomedicines) offer the potential of prolonging drug effects within the respiratory tract, thereby reducing the number of necessary inhalations. In the model of U46619-elicited PH, sildenafil and two sildenafil-loaded polymeric submicron particle formulations were evaluated for their pharmacodynamic and pharmacokinetic characteristics and acute tolerability. Lung-delivered sildenafil caused a selective dose-dependent decline of the pulmonary arterial pressure and vascular resistance. Compared to the transient pharmacodynamic effect observed for sildenafil, the same dose of nanoencapsulated sildenafil resulted in prolongation, but not augmentation, of the pulmonary vasodilatation. An extended pharmacokinetic profile was observed for nanoencapsulated sildenafil, and nanomedicines revealed no acute toxicity. The amplification of pulmonary vasodilatory response caused by nanoencapsulation of sildenafil offers an intriguing approach to ameliorate the therapy of PH. From the Clinical Editor: Pulmonary hypertension usually results in right heart failure long term. Current medical therapy includes the use of potent vasodilators such as sildenafil. In this article, the authors investigated the use of nanoencapsulated formulation for sustained delivery via inhalation route. An extended pharmacokinetic profile was seen for this nanoformulation with little side effects. It is hoped that clinical application of this would come to fruition soon.

Characterization of lung-delivered in-situ forming controlled release formulations.

OBJECTIVES: This study investigated the controlled drug release potential of formulations revealing temperature-induced sol-gel transition following administration to the respiratory tract. METHODS: Diverse sildenafil-containing aqueous poloxamer 407 preparations were evaluated for critical gelation temperature and rheological properties. The in-vitro drug release profiles of the in-situ forming formulations were studied in a Franz type cell, while the drug absorption characteristics were determined in an isolated lung model. Furthermore, the weight gain of isolated lungs was monitored and the bronchoalveolar lavage fluid was analysed for the total protein content. KEY FINDINGS: Poloxamer 407 solutions with concentrations of >12 wt.% revealed gelation upon temperature increase (>20°C). Compared with free sildenafil solution, sildenafil-containing polymer formulations showed a prolonged in-vitro drug release profile. Likewise, 17 and 21 wt.% of poloxamer 407 were characterized by a sustained sildenafil transfer from the lung into the perfusate. However, a 10 wt.% polymer solution displayed an immediate sildenafil absorption. Interestingly, increasing the poloxamer 407 concentration (21 and 17 vs. 10 wt.%) led to decreased organ weight gain kinetics and a lower total protein content found in the bronchoalveolar lavage fluid. CONCLUSIONS: In-situ forming controlled release hydrogels represent a viable approach for inhalative therapy.

Systematic aging of degradable nanosuspension ameliorates vibrating-mesh nebulizer performance.

CONTEXT: The process of vibrating-mesh nebulization is affected by sample physicochemical properties. Exemplary, electrolyte supplementation of diverse formulations facilitated the delivery of adequate aerosols for deep lung deposition. OBJECTIVE: This study addressed the impact of storage conditions of poly(lactide-co-glycolide) nanosuspension on aerosol properties when nebulized by the eFlow®rapid. MATERIALS AND METHODS: First, purified nanosuspensions were supplemented with electrolytes (i.e. sodium chloride, lactic and glycolic acid). Second, the degradable nanoparticles (NP) were incubated at different temperatures (i.e. 4, 22 and 36 °C) for up to two weeks. The effect of formulation supplementation and storage on aerosol characteristics was studied by laser diffraction and correlated with the sample conductivity. RESULTS AND DISCUSSION: Nebulization of purified nanosuspensions resulted in droplet diameters of >7.0 µm. However, electrolyte supplementation and storage, which led to an increase in sample conductivity (>10-20 µS/cm), were capable of providing smaller droplet diameters during vibrating-mesh nebulization (≤5.0 µm). No relevant change of NP properties (i.e. size, morphology, remaining mass and molecular weight of the employed polymer) was observed when incubated at 22 °C for two weeks. CONCLUSION: Sample aging is an alternative to electrolyte supplementation in order to ameliorate the aerosol characteristics of degradable NP formulations when nebulized by vibrating-mesh technology.

Biophysical inhibition of pulmonary surfactant function by polymeric nanoparticles: role of surfactant protein B and C.

The current study investigated the mechanisms involved in the process of biophysical inhibition of pulmonary surfactant by polymeric nanoparticles (NP). The minimal surface tension of diverse synthetic surfactants was monitored in the presence of bare and surface-decorated (i.e. poloxamer 407) sub-100 nm poly(lactide) NP. Moreover, the influence of NP on surfactant composition (i.e. surfactant protein (SP) content) was studied. Dose-elevations of SP advanced the biophysical activity of the tested surfactant preparation. Surfactant-associated protein C supplemented phospholipid mixtures (PLM-C) were shown to be more susceptible to biophysical inactivation by bare NP than phospholipid mixture supplemented with surfactant protein B (PLM-B) and PLM-B/C. Surfactant function was hindered owing to a drastic depletion of the SP content upon contact with bare NP. By contrast, surface-modified NP were capable of circumventing unwanted surfactant inhibition. Surfactant constitution influences the extent of biophysical inhibition by polymeric NP. Steric shielding of the NP surface minimizes unwanted NP-surfactant interactions, which represents an option for the development of surfactant-compatible nanomedicines.

Nebulization of active pharmaceutical ingredients with the eFlow(®) rapid: impact of formulation variables on aerodynamic characteristics.

Nebulization of active pharmaceutical ingredient (API) solutions is a well-established means to achieve pulmonary drug deposition. The current study identified the impact of formulation variables on the aerosolization performance of the eFlow(®) rapid with special respect to optimized lung application. API formulations (including excipient-supplemented samples) were investigated for physicochemical properties, then nebulized using vibrating-mesh technology. The generated aerosol clouds were analyzed by laser diffraction. Aerosol deposition characteristics in the human respiratory tract were estimated using an algebraic model. Remarkable effects on aerosolization performance [i.e., mass median aerodynamic diameter (MMAD)] of API solutions were obtained when the sample conductivity (by API concentration and type, sodium chloride addition) and dynamic viscosity (by application of sucrose and poly(ethylene glycol) 200) were elevated. A similar influence was observed for a decline in surface tension (by ethanol addition). Thus, a defined adjustment of formulation parameters allowed for a decrease of the MMAD from ∼ 8.0 µm to values as small as ∼ 3.5 µm. Consequently, the pattern and efficiency of aerosol deposition in the human respiratory tract were improved. In conclusion, identification of physicochemical variables and their way of influencing vibrating-mesh nebulization has been provided to deliver a platform for tailoring aerosol characteristics and thus, advancing pulmonary therapy.

Controlling the droplet size of formulations nebulized by vibrating-membrane technology.

Manipulation of aerosol characteristics is of special interest for pulmonary therapy, as a suitable particle size optimizes pulmonary deposition. The present study investigated the impact of formulation variables on the aerodynamic particle diameter (d(a)) when nebulized by vibrating-membrane technology. Membranes implemented in the Aeroneb® Pro and eFlow®rapid nebulizer revealed difference in metal composition and nozzle morphology as determined by energy dispersive X-ray measurements and scanning electron microscopy. Laser diffraction analysis of generated aerosol droplets identified the conductivity and dynamic viscosity of formulations as parameters with significant influence on the d(a) for both nebulizers. Accordingly, sample supplementation with particular excipients (conductivity: >50 µS/cm, dynamic viscosity: >1.5 mPa s) facilitated a reduction of the d(a) from ⩾8 µm, which is clearly in conflict with inhalative drug delivery, to respirable d(a) as small as ~3 µm. Overall, controlling the d(a) of formulations nebulized by vibrating-membrane technology seems to be technical feasible by an adequate adaption of samples' physicochemical properties. The Aeroneb® Pro and eFlow®rapid device are both qualified for the production of respirable aerosol clouds from specified formulations.

Polymer nanoparticle-based controlled pulmonary drug delivery.

The development of novel formulations for controlled pulmonary drug delivery purposes has gained remarkable interest in medicine. Although nanomedicine represents attractive concepts for the treatment of numerous systemic diseases, scant information is available on the controlled drug release characteristics of colloidal formulations following lung administration, which might be attributed to the lack of methods to follow their absorption and distribution behavior in the pulmonary environment.In this chapter, we describe the methods of preparation and characterization of drug-loaded polymeric nanoparticles prepared from biodegradable charge-modified branched polyesters, aerosolization of the nanosuspensions using a vibrating-mesh nebulizer, and evaluation of the pulmonary pharmacokinetics (i.e., absorption and distribution characteristics) of the nanoscale drug delivery vehicles following aerosol delivery to the airspace of an isolated lung model. The disclosed methodology may contribute to the design of advanced colloids for the treatment of respiratory disorders.

Biophysical inhibition of synthetic vs. naturally-derived pulmonary surfactant preparations by polymeric nanoparticles.

Reasonable suspicion has accumulated that inhaled nano-scale particulate matter influences the biophysical function of the pulmonary surfactant system. Hence, it is evident to provide novel insights into the extent and mechanisms of nanoparticle-surfactant interactions in order to facilitate the fabrication of safe nanomedicines suitable for pulmonary applications. Negatively- and positively-charged poly(styrene) nanoparticles (diameters of ~100nm) served as model carriers. Nanoparticles were incubated with several synthetic and naturally-derived pulmonary surfactants to characterize the sensitivity of each preparation to biophysical inactivation. Changes in surface properties (i.e. adsorption and dynamic surface tension behavior) were monitored in a pulsating bubble surfactometer. Both nanoparticle formulations revealed a dose-dependent influence on the biophysical behavior of all investigated pulmonary surfactants. However, the surfactant sensitivity towards inhibition depended on both the carrier type, where negatively-charged nanoparticles showed increased inactivation potency compared to their positively-charged counterparts, and surfactant composition. Among the surfactants tested, synthetic mixtures (i.e. phospholipids, phospholipids supplemented with surfactant protein B, and Venticute®) were more susceptible to surface-activity inhibition as the more complex naturally-derived preparations (i.e. Alveofact® and large surfactant aggregates isolated from rabbit bronchoalveolar lavage fluid). Overall, nanoparticle characteristics and surfactant constitution both influence the extent of biophysical inhibition of pulmonary surfactants.

Boosting the aerodynamic properties of vibrating-mesh nebulized polymeric nanosuspensions.

Pulmonary application of drug-loaded polymeric nanosuspensions is achieved by vibrating-mesh nebulizers, which allow for an output of intact nanocarriers from the nebulizer reservoir. However, adequate aerosol droplet sizes are a prerequisite for an efficient pulmonary deposition. The current study discloses experimental findings useful to optimize the aerodynamic characteristics of formulations atomized by the vibrating-mesh nebulizers Aeroneb(®) Pro and eFlow(®)rapid. Parameters with significant influence on the aerosol droplet diameter were identified by a statistical design analysis rating size results from laser diffraction. Subsequently, the effect of selected biocompatible solutes on the aerodynamic performance of nebulized formulations was studied and correlated with their physicochemical properties. Vibrating-mesh generated aerosols were significantly affected by the dynamic viscosity and conductivity of the applied formulation. Consequently, an increase in viscosity enhancer (sucrose and poly(ethylene glycol)) or electrolyte (NaCl and CaCl2) content caused the droplet diameter to decrease. Similarly, purified nanosuspensions revealed a considerable decline in aerosol particle size upon excipient addition. However, coating of polymeric nanoparticles with poloxamer and poly(vinyl alcohol) was necessary to avoid electrolyte-induced nanoparticle aggregation. Overall, the current study emphasizes that supplementation of nanosuspensions with biocompatible solutes is an excellent means to tailor the characteristics of aerosols generated by vibrating-mesh technology.

On the correlation of output rate and aerodynamic characteristics in vibrating-mesh-based aqueous aerosol delivery.

Aerosolization of aqueous formulations is of special interest for inhalative drug delivery, where an adequate nebulizer performance represents a prerequisite for improving pulmonary therapy. The present study investigated the interplay of output rate and aerodynamic characteristics of different excipient-based formulations and its impact on the atomization process by vibrating-mesh technology (i.e. eFlow(®)rapid). Output rate and aerodynamic characteristics were manipulated by both dynamic viscosity and conductivity of the applied formulation. Supplementation with sucrose and sodium chloride caused a decline (down to ∼0.2 g/min) and elevation (up to ∼1.0 g/min) of the nebulizer output rate, respectively. However, both excipients were capable of decreasing the aerodynamic diameter of produced aerosol droplets from >7.0 µm to values of ≤5.0 µm. Thus, the correlation of output rate and aerodynamic characteristics resulted in linear fits of opposite slopes (R(2)>0.85). Finally, the overall number of delivered aerosol droplets per time was almost constant for sucrose (≤1×10(8) droplets/s), while for sodium chloride a concentration-dependent increase was observed (up to ∼3×10(8) droplets/s). Overall, the current findings illustrated the influence of formulation parameters on the aerosolization process performed by vibrating-mesh technology. Moreover, concentration and charge distribution of aerosol populations supposedly modify the final characteristics of the delivered aerosols.

Correlation of drug release with pulmonary drug absorption profiles for nebulizable liposomal formulations.

Liposomes have attracted extensive attention as inhalative drug delivery vehicles. The preparation of tailored liposomal formulations (i.e. nebulization stability and controlled drug release profiles) would facilitate new perspectives for the treatment of pulmonary diseases. 5(6)-Carboxyfluorescein (CF)-loaded submicron liposomal formulations with varying phase transition temperatures were prepared from lipid blends in different molar ratios. Their physicochemical properties, in vitro dye release, stability to nebulization (Aeroneb Pro) and ex vivo pulmonary dye absorption and distribution characteristics were investigated. Phase transitions of liposomes were adjusted below and above body temperature (32.9-55.2 °C). The amount of CF released from liposomes in vitro correlated well with their membrane fluidity. An increase in phase transition temperature resulted in an extended dye release profile. All formulations revealed aerodynamic particle sizes of ∼4 µm with remarkable stability when nebulized by vibrating-mesh technology (percentage of encapsulated model drug ∼80%). Analogous to the release results observed in vitro, liposomal formulations revealing phase transitions above body temperature displayed an increased pulmonary CF retention in an ex vivo lung model. Consequently, an in vitro-ex vivo correlation was established, which demonstrated an excellent agreement of the dye release results with the absorption profiles observed in the biological system (R(2) ≥ 0.91). Overall, the concept of liposomal "phase transition release" is promising for controlled pulmonary drug delivery applications. The ex vivo technique enables a reliable determination of lung-specific pharmacokinetics of drug delivery vehicles, which enhances tailored carrier preparation and testing during early formulation development.

Following the concentration of polymeric nanoparticles during nebulization.

PURPOSE: Nebulization represents one strategy to achieve pulmonary deposition of biodegradable nanoparticles. Besides stability as a key requirement to maintain functionality, the output of nanoparticles from the nebulizer needs to be considered to facilitate an efficient pulmonary therapy. METHODS: Formulations nebulized by air-jet and vibrating-membrane technology were analyzed for their aerodynamic characteristics by laser diffraction. The nebulization stability of poly(D,L-lactide-co-glycolide) nanoparticles was assessed by dynamic light scattering. Moreover, several methods were employed to account for the shift in solute and NP reservoir concentration during nebulization. RESULTS: Regardless of the formulation or nebulizer used generated aerosols all showed aerodynamic characteristics suitable for deep lung deposition. However, nanoparticles were prone to aggregation and concentrated during air-jet nebulization. The particle concentration effect was significantly pronounced in comparison to molecular solutes under the same nebulization conditions, due to nanoparticle aggregation and subsequent particle remainder in the reservoir. In contrast, vibrating-membrane technology did not affect nanoparticle integrity and reservoir concentration during nebulization, as the unaffected submicron particles passed through the tapered holes of the actuated plate. CONCLUSIONS: Aggregation and concentration effects during air-jet nebulization emphasize that nanosuspensions should preferably be delivered with a suitable vibrating-membrane device in order to ensure an effective pulmonary application.

Micro-computed tomography imaging of composite nanoparticle distribution in the lung.

Nanomedicine comprises a significant potential to approach the therapy of severe diseases. Knowledge of nanoparticle behavior at the target site would contribute to the development of specialized tools for respiratory medicine. Here, we were interested in the potential of micro-computed tomography (µCT) imaging to monitor the pulmonary distribution of polymeric nanoparticles. Composite nanoparticles were analyzed for physicochemical properties, morphology and composition. µCT was employed to visualize the pulmonary distribution of composite nanoparticles in an ex vivo lung model. Employed composite nanoparticles were composed of poly(styrene) cores coated by a thin shell of colloidal iron oxide. Particles were mainly located in the interstitial space and associated with pulmonary cells, as observed by light microscopy. µCT detected enhanced X-ray opacities in the conducting (linear pattern) and respiratory airways (aciniform X-ray attenuations). In conclusion, multifunctional nanoparticles will prompt the development of novel therapeutic and diagnostic tools in respiratory medicine.

Impact of lyoprotectants for the stabilization of biodegradable nanoparticles on the performance of air-jet, ultrasonic, and vibrating-mesh nebulizers.

Strategies for further advancements in pulmonary drug delivery include the application of colloidal carriers. Freeze-drying in the presence of lyoprotectants is a valuable approach to improve long-term stability of biodegradable nanoparticles, but possibly constrains aerosol generation after powder rehydration, when employing traditional nebulizers. Here, we investigated the impact of lyoprotectants on both output and aerodynamic performance of air-jet, ultrasonic, and vibrating-mesh nebulization. Additionally, changes in formulation temperature and concentration were monitored, to estimate physicochemical alterations of formulations during nebulization. The stability of poly(d,l-lactide-co-glycolide) nanoparticles was maintained for lyoprotectant/nanoparticle ratios above 5/1. All nebulized formulations displayed suitable output and aerodynamic characteristics for peripheral lung deposition. Air-jet- and ultrasonic nebulization was associated with considerable temperature (~10°C) and concentration changes (up to 156%) of the reservoir fluid, which consequently, caused significant shifting of surface tension and viscosity. By contrast, vibrating-mesh nebulization caused marginal temperature increase (~5°C) with no apparent signs of concentration. Thus, the changing surface tension and viscosity were fitted employing Eötvös' rule and the Andrade equation (R(2)>0.98), allowing to predict the physicochemical properties of each formulation for prolonged nebulization periods. In particular, vibrating-mesh nebulization seems to be promising for aerosol application of rehydrated freeze-dried biodegradable nanoparticles to the respiratory tract.

Nebulization performance of biodegradable sildenafil-loaded nanoparticles using the Aeroneb Pro: formulation aspects and nanoparticle stability to nebulization.

Polymeric nanoparticles meet the increasing interest for drug delivery applications and hold great promise to improve controlled drug delivery to the lung. Here, we present a series of investigations that were carried out to understand the impact of formulation variables on the nebulization performance of novel biodegradable sildenafil-loaded nanoparticles designed for targeted aerosol therapy of life-threatening pulmonary arterial hypertension. Narrowly distributed poly(D,L-lactide-co-glycolide) nanoparticles (size: ∼200 nm) were prepared by a solvent evaporation technique using poly(vinyl alcohol) (PVA) as stabilizer. The aerodynamic and output characteristics using the Aeroneb Pro nebulizer correlated well with the dynamic viscosity of the employed fluids for nebulization. The nebulization performance was mainly affected by the amount of employed stabilizer, rather than by the applied nanoparticle concentration. Nanoparticles revealed physical stability against forces generated during aerosolization, what is attributed to the adsorbed PVA layer around the nanoparticles. Sildenafil was successfully encapsulated into nanoparticles (encapsulation efficiency: ∼80%). Size, size distribution and sildenafil content of nanoparticles were not affected by nebulization and the in vitro drug release profile demonstrated a sustained sildenafil release over ∼120 min. The current study suggests that the prepared sildenafil-loaded nanoparticles are a promising pharmaceutical for the therapy of pulmonary arterial hypertension.

Development of a biodegradable nanoparticle platform for sildenafil: formulation optimization by factorial design analysis combined with application of charge-modified branched polyesters.

Biodegradable nanoparticles have gained tremendous attraction as carriers for controlled drug delivery to the lung. Despite numerous advances in the field, e.g. development of suitable methods for pulmonary administration of polymeric nanoparticles, a sufficient association of the therapeutic agent with the carrier system as well as drug release in a controlled fashion remain considerable challenges. Hence, this study examines the optimization of biodegradable sildenafil-loaded nanoparticle formulations intended for aerosol treatment of pulmonary hypertension. A factorial design analysis was employed to identify the important experimental factors involved in the preparation of nanoparticles by the solvent evaporation technique. The effect of tailored charge-modified branched polyesters on drug loading and in vitro drug release from nanoparticles was also evaluated. Moreover, colloidal stability of obtained nanoparticles was assessed, and stabilization of nanoparticles by lyophilization was accomplished without additional excipients. Essential experimental factors were identified and optimized to allow the preparation of nanoparticles composed of linear polyesters with a sildenafil content of ~5 wt.%. The in vitro drug release profile from these nanoparticles demonstrated a sustained release of sildenafil over ~90 min. Application of charge-modified branched polyesters enhanced the drug content in nanoparticles and drug release profile, according to the charge-density present in the employed polymer. Accordingly an increase in drug loading by a factor of ~1.4, a prolonged drug release profile from nanoparticles over ~240 min was achieved. Sildenafil release from nanoparticles made of linear and charge-modified branched polyesters was governed by a diffusion process. The obtained drug diffusion coefficients were decreased as the charge-density present in the applied polymer was increased, which promotes the strategy to improve drug loading and release rates by electrostatic interactions between polymer and drug. In addition, nanoparticles showed high colloidal stability in different media of importance for pulmonary application and were successfully stabilized by lyophilization. In conclusion, optimization of the nanoparticle preparation process together with the application of tailored polymeric materials facilitated the synthesis of promising drug carriers for sildenafil that permit a novel treatment modality for severe pulmonary hypertension.

Characterization of novel spray-dried polymeric particles for controlled pulmonary drug delivery.

Numerous studies have addressed the controlled pulmonary drug delivery properties of colloidal particles. However, only scant information on the potential of spray-drying for submicron particle preparation is available. By exploiting the advantages of spray-drying, the characteristics of submicron particles can be optimized to meet the requirements necessary for lung application. Submicron particles were prepared from organic poly(d,l-lactide-co-glycolide) (PLGA) solutions, and composite particles were spray-dried from aqueous PLGA nanosuspensions. The feed concentration, as well as the spray-mesh diameter influenced the resulting particle sizes. Nanoparticles were virtually unaffected after spray-drying. The aerodynamic characteristics of both particle species revealed aerosol particle sizes suitable for deposition in the deep lungs (≤4µm). While the entrapped drug was released within ~90min from the composite particles, extensive drug retardation (~480min) was observed for PLGA particles spray-dried from organic solution. These results suggest that nanospray-drying is a convenient method to prepare submicron, controlled drug delivery vehicles useful for pulmonary application potentially allowing access to alveolar tissue.

The potential for inhaled treprostinil in the treatment of pulmonary arterial hypertension.

Inhaled treprostinil is a safe and well-tolerated approved pharmaceutical for the treatment of pulmonary arterial hypertension. In a series of open-label studies and in the pivotal trial with 253 patients, this long-acting prostacyclin analogue demonstrated pronounced pulmonary selectivity of vasodilatory effects, improved physical capacity and excellent tolerability and safety following aerosol administration. For efficient treatment, only four daily inhalations of treprostinil are necessary compared with six to nine in iloprost aerosol therapy. This review describes in detail the development of inhaled treprostinil, starting with intravenous epoprostenol followed by inhaled iloprost and subcutaneous treprostinil, all three representing well-established and widely approved prostanoid therapies for pulmonary hypertension. In order to circumvent the drawbacks of intravenous epoprostenol, stable prostacyclin analogues with similar pharmacological properties have been investigated. In addition, alternative routes of administration have been proposed and evaluated, mainly inhaled and subcutaneous delivery. The concept of inhaled treprostinil was to combine the pulmonary selectivity of an aerosolized vasodilator with the long-acting effects of a stable prostacyclin analogue. Pulmonary arterial hypertension remains, however, a severe, life-threatening disease, in spite of the enormous progress in specific drug therapy over the last decade. Therefore, further improvement of drug therapy will be essential, with clear potential for inhaled treprostinil: a reduction of inhalation frequency and duration would markedly improve quality of life and compliance, and a longer-lasting local prostanoid effect might further enhance the efficacy of inhaled treprostinil. The advantageous pharmacological properties of treprostinil offer the opportunity to establish a convenient metered dose inhaler as a delivery system, to combine inhaled treprostinil with available or future drugs for pulmonary arterial hypertension, or to develop sustained release formulations of treprostinil suitable for inhalation based on liposomes or biodegradable nanoparticles.

Amphiphilic, low molecular weight poly(ethylene imine) derivatives with enhanced stability for efficient pulmonary gene delivery.

BACKGROUND: Poly(ethylene imine) (PEI) is a widely used transfection reagent for mammalian cells, but in vivo application of PEI 25 kDa is restricted by its toxicity. Low molecular weight (LMW) PEI is less toxic, but also less efficient than its high molecular weight equivalent, and prone to aggregation. METHOD: A set of polymers was synthesized by coupling poly(ethylene glycol) (PEG) that contained either C(16/18) -chains (Cx-EO) or butyl-poly(propylene oxide)-co-poly(ethylene glycol) (ButPP). Critical micelle concentration (CMC) was determined for copolymers. Polyplexes were characterized by DNA binding ability, polyplex size and aggregation, hemolysis, and cytotoxicity. Transfection efficiency was tested in vitro and in vivo in mouse lungs. RESULTS: Copolymers formed stable complexes with DNA, and showed enhanced complex stability in isotonic solution for at least 1 h. CMC was determined for Cx-EO-PEI 4.7 and 8.3 at 0.0019 and 0.0037 mM, respectively; membrane activity in a haemolysis assay was demonstrated for ButPP-PEI: both factors possibly enhance endosomal escape effect after PEGylation. IC(50) values of all synthesized polymers were in the range 6-33 ng/ml. Transfection efficiency of unmodified LMW-PEIs was equivalent or better than that of PEI 25 as a result of aggregation in vitro. Cells treated with polyplexes of amphiphilic polymers showed reduced transfection compared to PEI 25. After instillation in mouse lungs, highest transfection efficiency was demonstrated with Cx-EO copolymer of lowest molecular weight PEI. CONCLUSIONS: A new set of polymers with low toxicity and high stability was synthesized, which contains promising candidates for pulmonary gene transfer, as documented by in vivo experiments in mice.